Nuclear receptors regulate a diverse set of physiological processes and represent attractive targets for therapeutic applications. Peroxisome proliferator-activated receptor alpha (PPAR1) are nuclear receptors that are activated by members of the N-acylethanolamine (NAE) family of lipids, which includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the endocannabinoid anandamide. PPAR1 receptors mediate the anti-inflammatory and anorexigenic effects of OEA and PEA and may therefore represent attractive therapeutic targets for the treatment of inflammation and pain. Due to their hydrophobicity, NAEs are unable to traverse the aqueous cytosol unassisted. It is currently not known how OEA or PEA navigate the cellular cytoplasm to reach nuclear PPAR1 receptors. Recently, we identified fatty acid binding proteins (FABPs) as intracellular carriers for the endocannabinoid anandamide. FABPs are cytosolic fatty acid trafficking proteins whose binding sites accommodate a broad range of lipophilic ligands and may likewise bind OEA and PEA. Their small size and ability to enter the nucleus renders FABPs as likely carriers for NAEs to PPAR1 receptors. We hypothesize that FABPs act as OEA and PEA transporters, and by affecting ligand availability, may regulate PPAR1 activity. The first aim of the current application is to determine whether FABPs transport NAEs to nuclear PPAR1 receptors. We will employ chemical and genetic approaches to inhibit FABP function and delineate the contribution of FABPs towards PPAR1 activity. This study will identify the first intranuclear NAE carriers and will shed insights into the regulation of NAE signaling. The other major goal of this application involves identifying the molecular target(s) of endocannabinoid/anandamide transport inhibitors. Owing to their lipophilic nature, NAEs have been proposed to passively diffuse through cellular membranes. However, carrier-mediated uptake of NAEs via a putative endocannabinoid membrane transporter has also been proposed. Despite lacking molecular evidence to substantiate its existence, hundreds of inhibitors targeting this putative transporter have been synthesized and continue to be actively used in the endocannabinoid research community. The lack of a bona fide cellular target for transport inhibitors raises questions about the specificity and validity of these compounds as research tools. We have recently shown that FABP inhibitors reduce the intracellular transport of anandamide, effects that are mimicked by transport inhibitors. The goal of this aim is to provide evidence that cytosolic FABPs are targets of endocannabinoid transport inhibitors. We will employ overexpression, knockdown, and direct binding approaches to demonstrate that these compounds interact with FABPs. Because transport inhibitors possess analgesic properties, identifying cellular targets for these compounds may unmask novel therapeutic targets and will clarify ambiguities associated with their use in drug abuse research. The overall outcome of this study will greatly enhance our understanding of NAE signaling and will ascribe novel functions to FABPs in endocannabinoid biology. PUBLIC HEALTH RELEVANCE: Modulation of N-acylethanolamine signaling offers promising therapeutic avenues for the treatment of various disorders. This proposal focuses upon characterizing proteins that regulate N-acylethanolamine signaling within the cell. The findings of this study may lead to future therapies for the treatment of pain and inflammation.